Insolubilization (or immobilization) of extracellular and of intracellular water soluble enzymes, that is, the fixation of catalytically active (native enzymes in an insolubilized, solid or quasisolid, shaped or technological structure has become of increasing technoligical and economic importance, as a means of rendering a particular enzyme product reusable in batch-type processes or suitable for a continuous mode of operation in a so-called enzyme reactor.
During recent years efforts have been devoted to the development of immobilization technology in the starch processing industry in connection with the manufacture of such products as high dextrose and high fructose syrups. Lately, the relative importance of the latter has been increasing in food and related industries, high fructose syrup being an advantageous substitute for sucrose and for (the less sweet) high dextrose syrup. Two additional types of sugar syrup based on starch hydrolysis having a substantial content of maltose, namely high maltose and high conversion syrup are being used to an increasing extent, particularly in the confectionary (hard candy) and canning industries, respectively.
The over-all industrial process of converting starch via high dextrose syrup into high fructose syrup comprises three consecutive steps, namely the starch thinning or liquefaction process to dextrins, catalyzed by acid and/or by a bacterial alpha-amylase, followed by saccharification to a high dextrose syrup and then the conversion of the latter into high fructose syrup, each process catalyzed by its specific enzyme, viz. amyloglucosidase and glucose isomerase, respectively. Whereas the most recent technological development has made continuous processes available for both the first and the third step of the above production sequence, industrial saccharification is still predominantly a batch-type process. The starting material of a high maltose type of syrup is also liquefied starch produced by either one of the methods just described. However, in this case the subsequent saccharification step resulting in a syrup of high maltose content (and usually containing relatively little glucose) is effected by means of a maltogenic amylase, preferably a fungal alpha-amylase. Like glucogenic saccharification, the corresponding maltogenic process is usually conducted batch-wise using the soluble enzyme.
Although the convenience of having at ones disposal a totally continuous production sequence is evident, the development of industrial enzyme reactor techniques involving the use of insolubilized amyloglucosidas in the saccharification step is only at a preliminary stage.
The prior art contains a number of references to the insolubilization of amyloglucosidase, or example by bonding the enzyme to insoluble inorganic or organic carriers. However, only few of these methods appear to have been developed beyond the laboratory bench scale. Among the methods that apparently have passed that stage specific mention should be made of the immobilization of amyloglucosidase by covalent bonding to porous particles of glass or ceramic material. In this connection reference is made to a recent article published by D. D. Lee et al. in "Die Starke", Vol. 27 (1975 pp 384-387.
In a continuous process in which a pilot plant size column was packed with this particular enzyme product and fed with solutions of commercially available dextrins, degrees of conversion to dextrose were attained which approached the minimal conversion of about 92 percent generally required in a batch saccharification process with soluble amyloglucosidase, although a conversion to above 93 percent is of course preferred. However, product analyses indicated that reversion reactions, i.e. amyloglucosidase catalyzed repolymerization of glucose into maltose, isomaltose and higher oligomers, occur to a greater extent in the column process that in the batch-type, free enzyme process.
The phenomenon of increased reversion encountered with an immobilized enzyme product of this type appears at least partly to be an inherent disadvantage of using porous enzyme carrier materials. Clearly, the porous structure contributes substantially to the total surface area lending itself to enzyme bonding and, consequently, to the maximal enzyme activity obtainable from the immobilized enzyme product.
However, the high concentration of amyloglucosidase attached to the carrier pore surface combined with a reduced diffusion rate within the pores inevitably results in locally high glucose concentrations, thus constituting favourable conditions for the promotion of enzyme processes having high K.sub.m -values. This is exactly the case with the undesirable reversion reactions, and particularly those in which isomaltose and isomaltriose are formed.
An insolubilized maltogenic alpha-amylase product, prepared by bonding the soluble enzyme to aminoethylcellulose by means of glutaraldehyde, is disclosed in U.S. Pat. No. 4,011,137 (Thompson et al.). However, according to this reference the intended use of the immobilized enzyme is limited to increasing the degree of conversion of dextrinized starch to glucose by means of a similarly immobilized amyloglucosidase preparation, the saccharification process being conducted by means of a mixture of the two immobilized enzymes. There is no demonstration whatsoever in the patent of the use of the immobilized maltogenic alpha-amylase for the production of a high maltose syrup.
An additional disadvantage of using a particulate immobilized saccharifying enzyme, in which the enzyme is bonded to a porous or reticular material which is essentially homogeneous throughout the particulate structure can be expected from the heterogeneity in molecular size of the dextrinized starch serving as substrate. All dextrins, whether produced by acid or by enzymatic hydrolysis of starch, contain substantial fractions of different glucose oligomers. Normally, the starch thinning process is stopped when the average chain length of the hydrolysate is in the range of from 6 to 10 glucose residues, and it is conceivable that steric hindrance may impede the diffusion of the larger dextrin molecules to enzyme sites embedded beyond a certain depth within the porous or reticular particle structure.
It is one object of the present invention to overcome or at least to mitigate the principal disadvantages encountered with the prior art immobilized saccharifying enzyme products described above, by providing an insolubilized product having process characteristics essentially similar to those of the soluble enzyme, irrespective of whether the immobilized product is used in a batch process or in a continuous mode of operation.
In the case of immobilized amyloglucosidase this requirement normally entails that a continuous saccharification process can be conducted under industrial process conditions (i.e. in terms of composition and flow rate of the dextrin feed) so as to yield a glucose concentration of at least 92 percent, the total concentration of disaccharides (maltose and isomaltose) and trisaccharides (mainly panose and isomaltotriose) not exceeding about 4 and 1 percent, respectively.
In the case of the immobilized maltogenic alpha-amylase the corresponding conversion parameters required would be: approximately 40-60 percent maltose, 25-35 percent maltotriose and, preferably, less than 10 percent glucose.
Since commercially available soluble saccharifying enzymes are relatively cheap and highly active, another preferable but not essential object of the present invention is the provision of economically favourable, insolubilized products. The pursuance of this goal necessitates the utilization of comparatively inexpensive, commercially available carrier and other auxiliary materials and, furthermore, the achievements of substantial recoveries of enzyme activity in the immobilization process. In addition, from a toxicological point of view all materials used should be acceptable for food processing purposes.
Briefly, these objects are attained according to the present invention which provides an immobilized saccharifying enzyme product in particulate form in which enzymatically inert, water-insoluble carrier particles are coated with a liquid-permeable, proteinaceous layer in which the saccharifying enzyme is immobilized by cross-linking with glutaraldehyde.
Attempts to prepare such insolubilized products in which the proteinaceous layer is composed of the glutaraldehyde cross-linked saccharifying enzyme alone proved unsuccessful in the sense that, irrespective of the choice of core material, they resulted in incompletely immobilized products from which the enzyme would rapidly leak away. Similar problems were encountered with a variety of water-insoluble particulate carrier materials of inorganic (for example mineral or ceramic) origin and with certain types of proteinaceous core material (such as granular soy protein), apparently because of an insufficient number of cross-link binding sites at the surface of such particles.